Lead optimization of 4-imidazolylflavans: New promising aromatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.03.043

Our previous studies have shown that several 7-substituted-4- imidazolylflavans are potent inhibitors of aromatase. These compounds were designed considering the anti-aromatase effect of some natural flavonoids and the importance of an azole ring for synthetic inhibitors such as letrozole or anastrozole towards binding to the heme iron of aromatase. In this study, we report the optimization of these lead compounds by the modulation of flavan A ring. The resulting 7,8-benzo-4-imidazolylflavans were tested in order to assess their ability to inhibit aromatase. Biological data concerning enantiomers obtained from the chiral separation of the racemate compound 4-imidazolyl-7-methoxyflavan are also presented.

Full text of DOI:10.1016/j.ejmech.2011.03.043

European Journal of Medicinal Chemistry 46 (2011) 2541e2545
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Lead optimization of 4-imidazolylflavans: New promising aromatase inhibitors
*
Samir Yahiaoui , Christelle Pouget, Jacques Buxeraud, Albert José Chulia, Catherine Fagnère
UPRES EA 4021 “Biomolécules et Thérapies anti-tumorales”, Faculté de Pharmacie, 2 rue du Docteur Marcland, 87025 LIMOGES cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Our previous studies have shown that several 7-substituted-4-imidazolylflavans are potent inhibitors of
aromatase. These compounds were designed considering the anti-aromatase effect of some natural
flavonoids and the importance of an azole ring for synthetic inhibitors such as letrozole or anastrozole
towards binding to the heme iron of aromatase. In this study, we report the optimization of these lead
compounds by the modulation of flavan A ring. The resulting 7,8-benzo-4-imidazolylflavans were tested
in order to assess their ability to inhibit aromatase. Biological data concerning enantiomers obtained
from the chiral separation of the racemate compound 4-imidazolyl-7-methoxyflavan are also presented.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 12 February 2011
Received in revised form
15 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Keywords:
Aromatase inhibitors
7,8-Benzo-4-imidazolylflavans
Enantiomers
Flavonoids
1. Introduction
for activity [9,10]; then, we demonstrated that hydroxyl groups are
also important on B ring at positions 30 and/or 40 [11]. Our inves-
Breast cancer is the most common cancer diagnosed in women
and represents the second leading cause of cancer-related deaths in
women [1]. Nearly one-third of all breast cancers and two-thirds of
postmenopausal breast cancers are hormone-dependent, estrogens
playing a critical role in cancer cell proliferation. Therefore, several
strategies were developed to remove the influence of these
hormones on tumour growth; one of the most promising strategies
is hormonotherapy based on aromatase inhibitors such as letrozole
and anastrozole [2,3]. Aromatase, which is a cytochrome P450
enzyme, is responsible for the final step of the estrogen biosyn-
thesis (i.e. the conversion of androgens to estrogens) and therefore
is considered as a particularly attractive target for inhibition in the
treatment of hormone-dependent breast cancer [4].
In addition to the large number of aromatase inhibitors that
have arisen from medicinal chemistry efforts, some natural prod-
ucts such as flavonoids display an anti-aromatase activity [5,6].
Therefore, some research groups are pursuing drug discovery
efforts exploiting flavonoids, including isoflavonoids [7,8], as core
templates for novel aromatase inhibitors; with this aim, we have
undertaken the modulation of A, B and C rings (see Scheme 1) of
the flavan skeleton. Thus, some structureeactivity relationships
were evidenced through our different studies. First, a hydroxyl or
a methoxy group on A ring at position 7 was found to be essential
tigations for the C ring modulation of flavan skeleton were directed
towards the replacement of the flavanone 4-keto function by an
azole ring (imidazolyl or triazolyl moiety) [12e14]. The introduc-
tion of an imidazolyl moiety resulted in 4-imidazolylflavans which
are the most active molecules among the whole products we
synthesized; some of them demonstrated a high potential against
aromatase, exhibiting IC50 (40 nM) not far from that of letrozole
(IC50 ¼ 18 nM).
In order to optimize these lead compounds and to increase their
interesting potency, we report in the course of the present paper,
the synthesis of a new class of 4-imidazolylflavans substituted by
a benzo ring at positions C-7 and C-8 (Scheme 1). This A ring
modulation is based on the results of several studies which have
shown that the presence of a benzo group at C-7 and C-8 on flavone
skeleton is crucial for enhanced inhibitory activity [15,16]. Our
previous work dealing with 7,8-benzoflavanones [17], confirmed
this structureeactivity relationship, the latter compounds being
with the 8-prenylnaringenin [18,19], the most potent of the whole
flavanones reported in the literature. In the present work, the
synthesis of the non-substituted 7,8-benzo-4-imidazolylflavan was
performed and cyano and hydroxyl groups were also introduced at
the 40 position of the B ring. First, the cyano group is common in
several aromatase inhibitors such as anastrozole, letrozole or pyr-
azole based heterocycles [20]; in the azole family of aromatase
inhibitors (letrozole, indole derivatives [21]), the presence of
a para-cyano phenyl group is known to be important for activity.
Secondly, we have previously demonstrated, that a 40-hydroxy
* Corresponding author. Tel.: þ33 555 435 996; fax: þ33 555 435 801.
E-mail address: samir.yahiaoui@unilim.fr (S. Yahiaoui).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.043

2542
S. Yahiaoui et al. / European Journal of Medicinal Chemistry 46 (2011) 2541e2545
alkaline medium resulting from the presence of NaBH4 was
5''
3'
R
2'
1'
6''
8
responsible for the isomerization into the corresponding
20-hydroxychalcones, as evidenced by NMR analysis. This reaction,
except for the 40-hydroxy derivative, was stereoselective since it led
only to 2,4-cis-7,8-benzoflavan-4-ols. The hydride attack on the
opposite side from the 2-phenyl group is responsible for this
stereochemistry. The action of NaBH4 on 7,8-benzo-40-hydroxy-
flavanone led to a mixture of 2,4-cis and 2,4-trans isomers in a 1:1
ratio, as previously described for the reduction of 40-hydroxy-7-
methoxyflavanone and 40,7-dihydroxyflavanone [13]. However,
these two isomers were not separated.
4''
3''
4'
5'
B
O 1
C
7
2
6'
A
2a : R= H
2b : R= CN
2c : R = OH
6
3
4
5
N
5-imid
2-imid
N
4-imid
Scheme 1. Structure of the 7,8-benzo-4-imidazolylflavans 2a, 2b and 2c.
Excellent yields of these products were obtained (around 80%);
nevertheless, flavan-4-ols deteriorate if not carefully handled and
should be stored at low temperature. The instability may result
from their possible intramolecular dehydration into flav-3-enes, as
previously observed [22].
substitution was responsible for an increase in aromatase inhibition
in the case of a 4-imidazolyl-7-methoxyflavan [13].
These original 7,8-benzo-4-imidazolylflavans were obtained
using the strategy described for the synthesis of previous 4-imi-
dazolylflavans, involving chalcones, flavanones and flavan-4-ols as
synthetic intermediates [12]. The synthesis and the characteriza-
tion of the target compounds and of the 7,8-benzoflavan-4-ol
intermediates are reported as well as the results of the biological
evaluation against aromatase.
2.1.3. 7,8-Benzo-4-imidazolylflavans
In our previous studies, the substitution reaction on flavan-4-
ols, using 1,10-carbonyldiimidazole, led only to the 2,4-trans-4-
imidazolylflavans [12,13]; however, in this work, the action of the
same reagent on the 2,4-cis-7,8-benzoflavan-4-ols led also, for the
first time, to the corresponding 2,4-cis-4-imidazolylflavans for
compounds 2b (40-cyano) and 2c (40-hydroxy). Separation of the
two stereoisomers of these 7,8-benzo-4-imidazolylflavans was
undertaken but compound 2b was the only one from which 2,4-cis
and 2,4-trans isomers were obtained as pure molecules (1:4 ratio
between the 2,4-cis and the 2,4-trans isomer).
2. Results and discussion
2.1. Chemistry
2.1.1. General procedure of synthesis
2.2. Aromatase inhibitory activity
In this work, three 7,8-benzo-4-imidazolylflavans were
synthesized: the 7,8-benzo-4-imidazolylflavan (2a), the 40-cyano
derivative (2b) and the 40-hydroxyl derivative (2c). The synthesis of
these compounds is a multi-step process requiring three classes of
intermediate products: 30,40-benzo-20-hydroxychalcones, 7,8-ben-
zoflavanones and 7,8-benzoflavan-4-ols. The synthesis pathway is
depicted in Scheme 2. Benzochalcones and 7,8-benzoflavanones
were described elsewhere [17].
The pharmacological data - IC50 values and the potencies (RP)
relative to aminoglutethimide which is the first aromatase
inhibitor used clinically
- are summarized in Table 1. For
compound 2a, the 2,4-trans isomer was the only obtained; for
derivative 2b, the two isomers 2,4-cis and 2,4-trans were sepa-
rated and evaluated as pure molecules. A 50/50 mixture of these
two isomers was also made for measuring its inhibitory effect.
Then, for compound 2c, anti-aromatase activity was evaluated for
a 50/50 mixture of the two isomers. Previous results concerning
7,8-benzoflavanones and 2,4-trans-4-imidazolyl-7-methoxy-
flavans are also reported (Table 1).
2.1.2. 7,8-Benzoflavan-4-ols
Reduction of the 7,8-benzoflavanones was carried out at low
temperature (6 ꢀC) using NaBH4; indeed, at room temperature, the
R
R
OH
OH
Ba(OH)2
+
H
CH3
EtOH
H2SO4/MeOH
O
O
O
3',4'-benzo-2'-hydroxychalcones
R
O
R
R
O
O
7,8-benzoflavanones
NaBH4
EtOH
O
1,1'-carbonyldiimidazole
THF
N
N
OH
a
H
b
c
7,8-benzo-4-imidazolylflavans
7,8-benzoflavan-ols
R
CN OH
2a-2c
1a-1c
Scheme 2. Synthesis pathway for 7,8-benzo-4-imidazolylflavans (2ae2c).

S. Yahiaoui et al. / European Journal of Medicinal Chemistry 46 (2011) 2541e2545
2543
Table 1
Aromatase inhibitory activity of 7,8-benzo-4-imidazolylflavans (2ae2c) *The relative potency (RP) was calculated from the ratio of IC50 values for the tested compounds to that
measured for aminoglutethimide (AG) (IC50 ¼ 5.8 M).
m
B ring substitution
7,8-benzo flavanones
4-imidazolylflavans
7,8-benzo
2,4-transe7-methoxy
IC50
4.3
1.54
(m
M)
Compound
stereochemistry
IC50
(
mM)
RP/AG*
RP/AG*
not substituted
2a
2b
2,4-trans
2,4-cis
2,4-trans
0.036
0.534
0.034
0.078
0.032
161
11
170
74
64
40eCN
39
2,4-transþ2,4-cis (1:1)
40eOH
0.63
2c
2,4-transþ2,4-cis (1:1)
181
130
First, the derivatives show high inhibitory activity against
aromatase with IC50 values from 0.03 to 0.53 M and are more
potent than aminoglutethimide (IC50 ¼ 5.8 M) exhibiting rela-
the 40-hydroxy substitution on 7,8-benzoflavanone [17], and on
4-imidazolyl-7-methoxyflavan [13], was shown to increase aro-
matase inhibition. Comparing the activities of the 50/50 mixture
for compounds 2b and 2c, it appears that an additional
40-hydroxyl group is more favorable to the anti-aromatase effect;
thus, the 40-hydroxy derivative is 2.5 times more potent than the
40-cyano analog. Certainly, the separation between the two
isomers (2,4-trans and 2,4-cis) of compound 2c was not per-
formed but considering that a 2,4-trans isomer is twice more
potent than a 50/50 mixture of the two isomers, a great IC50
value for the 2,4-trans isomer of compound 2c could be expected.
Thus, this 2,4-trans compound could be, in vitro, as active as
m
m
tive potencies (RP) from 11 to 180. Comparing the target
compounds with their corresponding flavanones, it became
apparent that replacement of the carbonyl function by an imi-
dazolyl group lead to a strong increase in enzyme inhibition.
Thus, the 2,4-trans-7,8-benzo-4-imidazolylflavan 2a is 120 times
more potent than the 7,8-benzoflavanone. This result is in
agreement with those previously described for 7-hydroxy-, and
7-methoxyflavanones [12].
Besides, comparing the activities of 7,8-benzo-4-imidazolyl-
flavans with those of their 7-methoxy counterparts, it is evident
that a benzo ring at C-7 and C-8 positions of 4-imidazolylflavan
skeleton is more attractive than a 7-methoxy group for an
enhanced aromatase inhibition [13]. Thus, the 7,8-benzo-4-imi-
dazolylflavans were found to be more potent than 7-methoxy
analogs. These findings reinforce the results of many studies
dealing with flavonoid inhibitory effect, like 7,8-benzoflavanones
[17], or 7,8-benzoflavone [5,15,16], and demonstrating that an
additional 7,8-benzo ring is of great interest to obtain potent
aromatase inhibitors.
letrozole (IC50 ¼ 0.018
mM), which is used as the first-line
therapy for metastatic breast cancer. The introduction of this
40-hydroxyl group on B ring of 7,8-benzo-4-imidazolylflavan
scaffold seems very important to reinforce the interaction
between the inhibitor and the aromatase active site. So, our prior
interest is now to carry out further work to separate the two
isomers of compound 2c in order to obtain the 2,4-trans isomer
as a pure molecule and to confirm the great influence of
stereochemistry on biological activity.
It is also of importance to consider that our 4-imidazolylflavans
were always tested as racemate compounds; to further investigate
the influence of stereochemistry on biological activity, the first
active 2,4-trans-4-imidazolyl-7-methoxyflavan that we synthe-
sized, was separated into its enantiomers (2S,4R and 2R,4S)
(Scheme 3) using chiral HPLC (ChiralpakÒAD) with hexane/ethanol
80/20 as an eluent. Thus, pure enantiomers of a 2,4-trans-4-imi-
dazolylflavan were tested, for the first time, against aromatase
(Table 2). The (þ) enantiomer was found to be a highly potent
inhibitor, being 1.5 and 10 times more active than its racemate and
the (ꢁ) enantiomer, respectively. This result confirms the impor-
tance of the chiral center configuration towards aromatase inhibi-
tion, as previously observed for vorozole [23] and for indole
derivatives [21].
Then, for the first time,
obtained and evaluated for anti-aromatase effect.
a
2,4-cis-4-imidazolylflavan was
marked
A
difference of activity was observed between the two isomers (2,4-
trans and 2,4-cis) of compound 2b; 2,4-cis isomer was found 15
times less potent than the 2,4-trans derivative. A 50/50 mixture of
the two isomers was made and it was found to be twice less potent
than the 2,4-trans isomer.
Finally, the influence of the 40-substituent (B ring) on the
aromatase inhibitory activity was investigated. By comparing
activity of 2,4-trans isomers for compounds 2a and 2b, it appears
that a cyano group at position 40 do not increase the aromatase
inhibition as previously noticed for the 4-imidazolyl-7-methox-
yflavan derivatives [13]. On the contrary, an additional 40-cyano
group on the 7,8-benzoflavanone skeleton was previously found
to enhance the anti-aromatase effect [17]. These findings may
suggest a different mode of binding to the active site of the
aromatase between flavanones and 4-imidazolylflavans. Then, it
was important to study the influence of a 40-hydroxyl group since
3. Conclusion
With the aim to design new leads for aromatase inhibition, we
are pursuing the modulation of flavan skeleton and we synthesized
novel 7,8-benzo-4-imidazolylflavans which were tested against
aromatase, exhibiting a higher inhibitory activity than 4-imida-
zolyl-7-methoxyflavans. Thus, the 2,4-trans isomer of 7,8-benzo-40-
cyano-4-imidazolylflavan 2b is only twice less active than letrozole.
O
O
N
O
O
N
Table 2
Aromatase inhibitory activity of 2,4-trans-4-imidazolyl-7-methoxyflavan.
N
Compound
IC50 M)
RP/AG
racemate
(þ) enantiomer
0.060
97
(ꢁ) enantiomer
0.600
10
N
2R,4S
2S,4R
(
m
0.091
64
Scheme 3. Structure of the 2,4-trans-4-imidazolyl-7-methoxyflavan enantiomers.

2544
S. Yahiaoui et al. / European Journal of Medicinal Chemistry 46 (2011) 2541e2545
Besides, a 2,4-cis isomer was obtained for the first time and was
found to be less potent than the corresponding 2,4-trans isomer,
which demonstrates the great influence of stereochemistry on
biological effect. So, it is of interest to work further to the separation
of the 2,4-cis and 2,4-trans isomers of compound 2c since the 50/50
mixture was evaluated to be only twice less active than letrozole.
We have also demonstrated for the first time the difference of
activity between the two enantiomers (2S,4R and 2R,4S) of a 2,4-
trans-4-imidazolylflavan. Therefore, a next step in this work is the
chiral separation of all these racemic compounds, in order to
enhance anti-aromatase effect by testing enantiomerically pure
compounds. Further experiments directed towards X-ray crystal-
lographic and molecular modeling studies, are currently under-
going, considering that 7,8-benzo-4-imidazolylflavans could
probably result in novel drug candidates for the treatment of
hormone-dependent breast cancer.
dt, J ¼ 2.6 and 14.4 Hz, H-3eq),
14.4 Hz, H-3ax),
5.18 (1H, dd, J ¼ 2.4 and 11.0 Hz, H-2),
br t, J ¼ 3.6 Hz, H-4), 6.70 (1H, br s, H-4imid), 7.12 (1H, br s,
H-5imid), 7.35 to 7.59 (9H, m, H-6;
7,14 (1H, d, J ¼ 8.5 Hz, H-5),
d
2.59 (1H, ddd, J ¼ 4.6; 11.0 and
d
d
5.49 (1H,
d
d
d
d
d
H-20; H-30; H-40; H-50; H-60; H-400; H-500 and H-2imid),
d 7.82 (1H, br
d, J ¼ 7.5 Hz, H-300),
d
8.20 (1H, br d, J ¼ 8.0 Hz, H-600). 13C NMR
(100 MHz; CDCl3):
d 38.5 (C-3), d 51.6 (C-4), d 73.5 (C-2), d 110.9
(C-4a),
d
d
d
118.7 (C-4imid),
126.0 (C-20 and C-60),
127.6 (C-300), 128.4 (C-40),
134.7 (C-7),
d
121.2 (C-6),
d
d
122.3 (C-600),
125.2 (C-8),
126.5 (C-5), d
127.5 (C-400),
d
d
126.1 (C-500),
d
d
128.8 (C-30 and C-50),
d
d
129.7
151.3
(C-5imid),
d
d
137.0 (C-2imid),
d
139.8 (C-10),
(C-8a). HRMS (ESIþ) calculated for C22H19N2O (M þ H)þ 327.1497,
found 327.1506.
4.3.2. 40-cyano derivatives
2,4-cis-7,8-Benzo-40-cyano-flavan-4-ol (1b) was obtained in an
81% yield, as a white amorphous solid; mp 122 ꢀC. UV in MeOH
4. Experimental
lmax: 221, 237, 294, 323 nm 1H NMR (400 MHz; CD3OD) :
d
2.09 (1H,
2.58 (1H, ddd, J ¼ 1.9; 6.4
5.22 (1H, dd, J ¼ 6.4 and 10.3 Hz, H-4), 5.46
(1H, dd, J ¼ 1.7 and 11.9 Hz, H-2), 7.42 (1H, m, H-5”), 7.45 (1H, m,
7.60 (1H, d, J ¼ 8.6 Hz, H-5),
7.78 (1H, m, H-300),
7.81
8.14 (1H, br d, J ¼ 8.2 Hz, H-
40.9 (C-3), 66.0 (C-4), 77.8 (C-
121.2 (C-4a), 121.5 (C-6), 122.8
126.5 (C-5”), 127.4 (C-4”),
133.7 (C-30 and C-50),
135.5
ddd, J ¼ 10.3; 12.0 and 12.9 Hz, H-3ax),
d
4.1. General experimental procedures
and 13.0 Hz, H-3eq),
d
d
d
d
Compounds were purified by preparative thin layer chroma-
tography on MachereyeNagel silica gel. NMR spectra were recor-
ded on a Bruker 400 MHz spectrometer with Me4Si as an internal
standard. Mass spectrometric (ESI-TOF) analysis was carried out on
a Waters Alliance system equipped with electrospray interface.
H-4”),
d
d
7.46 (1H, d, J ¼ 8.8 Hz, H-6),
d
7.77 (2H, d, J ¼ 8.3 Hz, H-20 and H-60),
d
d
(2H, d, J ¼ 8.5 Hz, H-30 and H-50),
d
6”).13C NMR (100 MHz; CD3OD) :
d
d
d
d
2),
(C-6”),
d
d
112.7 (C-40),
125.7 (C-5),
128.0 (C-20 and C-60),
d
119.7 (CN),
d
d
d
d
126.1 (C-8),
d
d
d
4.2. Synthesis
d
d
128.6 (C-3”),
150.3 (C-8a). HRMS (ESIþ) calculated for
d
(C-7),
d
148.2 (C-10),
To a solution of 7,8-benzoflavanone in ethanol was added
sodium borohydride (5 eq). The mixture was kept under nitrogen
atmosphere at a temperature of 6 ꢀC for 24e72 h, then evaporated
under reduced pressure. Water was added, the pH was adjusted to
6 using 1M HCl and the resulting solution was extracted with
chloroform. The organic layers were washed with water, dried over
Na2SO4 and evaporated. Preparative TLC on silica gel afforded pure
flavan-4-ols.
To a solution of flavan-4-ol in anhydrous THF was added
1,10-carbonyldiimidazole (4eq). The mixture was kept under
nitrogen atmosphere at room temperature for 20e48 h. Subse-
quently, it was evaporated under reduced pressure. The residue was
dissolved in chloroform and the organic layer was washed with
water, dried over Na2SO4 and evaporated. The resulting 4-imida-
zolylflavans were purified via preparative TLC on silica gel.
C20H15NO2Na (M þ Na)þ 324.1000, found 324.1005.
4.3.2.1. 7,8-Benzo-40-cyano-4-imidazolylflavan (2b). 2,4-cis isomer :
was obtained in a 7% yield, as a white amorphous solid; mp
220 ꢀC.UV in MeOH lmax: 221, 242, 282, 323 nm 1H NMR (400 MHz;
CDCl3):
J ¼ 1.8; 6.3 and 13.5 Hz, H-3eq),
5.88 (1H, dd, J ¼ 6.3 and 11.2 Hz, H-4),
H-5), 6.85 (1H, br s, H-4imid), 7.12 (1H, br s, H-5imid),
d, J ¼ 8.6 Hz, H-6),
d
2.54 (1H, dt, J ¼ 11.6 and 13.2 Hz, H-3ax),
5.53 (1H, br d, J ¼ 10.5 Hz; H-2),
6.82 (1H, d, J ¼ 8.6 Hz,
7.40 (1H,
d 2.77 (1H, ddd,
d
d
d
d
d
d
d
7.53 (1H, m, H-500),
d
7.55 (1H, m, H-400),
d
d
7.69
7.77
(1H, br s, H-2imid),
d
7.71 (2H, d, J ¼ 8.3 Hz, H-20and H-60),
(2H, d, J ¼ 8.4 Hz, H-30 and H-50),
d
7.80 (1H, m, H-300),
d
8.25 (1H, br
53.3
117.5 (C-4imid),
123.7 (C-5), 124.8 (C-8),
127.3 (C-400), 127.7 (C-300),
134.3 (C-7), 137.2
d, J ¼ 8.0 Hz, H-600). 13C NMR (100 MHz; CDCl3) :
d 39.1 (C-3), d
(C-4),
d
76.6 (C-2),
d
112.4 (C-40),
d
114.3 (C-4a), d
d
d
d
118.5 (CN),
126.3 (C-500),
130.2 (C-5imid),
d
121.7 (C-6),
d
d
121.9 (C-600),
d
d
126.6 (C-20 and C-60),
d
d
4.3. Characterization of compounds
d
132.7 (C-30 and C-50),
d
d
(C-2imid),
d
144.9 (C-10),
d
150.0 (C-8a). HRMS (ESIþ) calculated for
4.3.1. Non-substituted derivatives
C23H18N3O (M þ H)þ 352.1450, found 352.1460.
2,4-cis-7,8-Benzoflavan-4-ol (1a) was obtained in an 81% yield,
2,4-trans isomer : was obtained in a 30% yield, as a white
amorphous solid; mp 216 ꢀC.UV in MeOH lmax: 222, 241, 281,
as a white amorphous solid; mp 168 ꢀC. UV in MeOH lmax: 218, 239,
296, 324 nm.1H NMR (400 MHz; CDCl3):
d
2.25 (1H, ddd, J ¼ 9.9;
323 nm 1H NMR (400 MHz; CDCl3) :
d
2.48 to
5.22 (1H, dd, J ¼ 4.7 and 9.2 Hz, H-2),
(1H, br t, J ¼ 3,6 Hz, H-4), 6.97 (1H, br s, H-4imid), 7.15 (1H, br
s, H-5imid), 7.49 (1H, d, J ¼ 8.5 Hz,
7,17 (1H, d, J ¼ 8.5 Hz, H-5),
d
2.57 (2H, m, H-
11.3 and 13.2 Hz, H-3ax),
3eq),
11.3 Hz, H-2),
d
2.66 (1H, ddd, J ¼ 2.1; 6.4 and 13.3 Hz, H-
3eq and H-3ax),
d
d
5.51
d
5.20 (1H, br t, J ¼ 7.7 Hz, H-4),
d
5.35 (1H, dd, J ¼ 1.7 and
d
d
d
7.36 (1H, dt, J ¼ 2.4 and 7.2 Hz, H-40),
d
7.41e7.53
d
d
(5H, m, H-6; H-400; H-500; H30 and H50),
d
7.52 (2H, d, J ¼ 7.2 Hz, H-20
H-6),
d
7.51 (1H, br s, H-2imid),
d
7.57 (1H, m, H-500),
d
7.59 (2H, d,
and H-60),
d
7.58 (1H, d, J ¼ 8.5 Hz, H-5),
d
7.77 (1H, br d, J ¼ 7.9 Hz,
J ¼ 8.2 Hz, H-20 and H-60),
d d 7.73 (2H, d,
7.60 (1H, m, H-400),
H-300),
d
8.23 (1H, br d, J ¼ 8.6 Hz, H-600). 13C NMR (100 MHz; CDCl3):
J ¼ 8.4 Hz, H-30 and H-50),
d
7.84 (1H, br d, J ¼ 8.4 Hz, H-300),
d
8.31
38.7 (C-
118.5
124.9 (C-8),
127.7 (C-
132.7 (C-30 and C-50),
d
d
40.0 (C-3),
d
65.8 (C-4),
124.3 (C-5),
126.5 (C-400), 127.4 (C-300),
134.1 (C-7),
140.7 (C-10),
d
76.9 (C-2),
d
119.1 (C-4a),
d
120.4 (C-6),
125,9 (C-
128.6 (C-30
(1H, br d, J ¼ 8.2 Hz, H-600). 13C NMR (100 MHz; CDCl3):
d
122.2 (C-600),
d
d
124.8 (C-8),
d
125.5 (C-500),
d
3),
d
51.1 (C-4),
d
72.7 (C-2),
d
110.8 (C-4a),
d
112.3 (C-40),
d
20 and C-60),
d
d
128.1 (C-40),
d
(C-4imid),
d
d
118.5 (CN),
126.5 (C-5),
127.8 (C-300),
130.0 (C-5imid),
d
121.7 (C-6),
d
122.0 (C-600),
d
and C-50),
d
d
d
149.6 (C-8a). HRMS (ESIþ)
126.3 (C-500),
d
d d
126.7 (C-20 and H-60),
calculated for C19H16O2Na (M þ Na)þ 299.1048, found 299.1056.
2,4-trans-7,8-Benzo-4-imidazolylflavan (2a) was obtained in
a 21% yield, as a white amorphous solid; mp 170 ꢀC.UV in MeOH
400),
d
d
d
d
134.7 (C-7), d 136.9 (C-2imid), d d 150.6 (C-8a).
145.1 (C-10),
HRMS (ESIþ) calculated for C23H18N3O (M þ H)þ 352.1450,
lmax: 216, 236, 291, 323 nm 1H NMR (400 MHz; CDCl3):
d
2.50 (1H,
found352.1449.

S. Yahiaoui et al. / European Journal of Medicinal Chemistry 46 (2011) 2541e2545
2545
4.3.3. 40-hydroxy derivatives
7,8-Benzo-40-hydroxy-flavan-4-ol (1c) was obtained in an 81%
yield, as a white amorphous solid; mp 190 ꢀC. UV in MeOH lmax
4.4. Aromatase assay
:
Inhibitory activity of the tested compounds on aromatase was
evaluated in vitro following Le Bail et al. procedure [9], using human
placental microsomes and [1,2,6,7-3H] androstenedione as
substrate. All enzymatic experiments were performed at 37 ꢀC in
0.1 M phosphate buffer. The final incubation mixture (1 mL) con-
tained 3 mM glucose-6-phosphate, 0.5 mM NADP and 0.1 unit
glucose-6-phosphate dehydrogenase in the presence or absence of
inhibitor. The steroids were extracted from the incubation mixture,
identified and quantified using HPLC and Packard Flow Scintillation
Analyzer 150 TR. Inhibitory activity of these compounds at five
appropriate concentrations was expressed as percent inhibition of
the aromatization of androstenedione which had an initial
concentration of 40 nM. In order to determine the IC50 value, all
experiments were performed in duplicate. The IC50 value of the
221, 238, 284, 322 nm. HRMS (ESIþ) calculated for C19H16O3Na
(M þ Na)þ 315.0997, found 315.1008.
2,4-cis isomer : 1H NMR (400 MHz; CD3OD) :
d 2.17 (1H, ddd,
J ¼ 10.7; 12.6 and 13.0 Hz, H-3ax),
d
2.49 (1H, ddd, J ¼ 1.7; 6.4 and
13.0 Hz, H-3 eq),
d
5.19 (1H, dd, J ¼ 6.5 and 10.5 Hz, H-4),
d 5.23 (1H,
dd, J ¼ 1.2 and 12.6 Hz, H-2),
d
6.85 (2H, d, J ¼ 8.5 Hz, H-30 and H-50),
d
d
d
7.37 (1H, m, H-500),
d
7.38 (2H, d, J ¼ 8.4 Hz, H-20 and C-60),
7.38e7.47 (2H, m, H-6 and H-400),
d
7.59 (1H, d, J ¼ 8.6 Hz, H-5),
7.74 (1H, br d, J ¼ 7.3 Hz, H-300),
d
8.09 (1H, br d, J ¼ 8.0 Hz, H-600).
13C NMR (100 MHz; CD3OD) :
d
41.0 (C-3),
118.5 (C-4a),
126.6 (C-8),
133.6 (C-10),
d 66.6 (C-4), d 78.7 (C-2),
d
d
116.3 (C-30 and C-50),
d
d
120.9 (C-6),
127.2 (C-400),
128.4
135.5 (C-7), d 151.1
d
123.1 (C-600),
125.7 (C-5),
d
126.2 (C-500),
d
d
d
(C-300),
(C-8a),
d
128.8 (C-20 and C-60),
158.5(C-40).
d
d
d
reference molecules, aminoglutethimide and letrozole are 5.8
and 0.018 M respectively.
mM
2,4-trans isomer : 1H NMR (400 MHz; CD3OD) :
d
2,19 (1H, ddd,
m
J ¼ 3.5; 11.4 and 15.0 Hz, H-3ax),
d
2.26 (1H, dt, J ¼ 2.6 and 14.2 Hz,
H-3 eq),
d
4.85 (1H, m, H-4),
d
5.27 (1H, dd, J ¼ 2.8 and 11.5 Hz, H-2),
Acknowledgments
d
6.86 (2H, d, J ¼ 8.5 Hz, H-30 and H-50),
d
7.38 (2H, d, J ¼ 8.4 Hz, H-20
and H-60),
d d 7.45 (1H, m,
7.38e7.43 (3H, m, H-5; H-6 and H-500),
The authors are grateful to the CNRS central service of analyses,
Vernaison, France, for recording the high resolution mass spec-
trometry; to Yves Champavier, Service Commun de RMN, Uni-
versité de Limoges, for recording the NMR spectra and to Nicolas
Vanthuyne, ISM2, Université de Marseille, for achievement of the
chiral separation.
H-400),
d
7.76 (1H, br d, J ¼ 7.6 Hz, H-300),
d
8.17 (1H, br d, J ¼ 7.7 Hz,
64.7 (C-4), 74.7
121.1 (C-4a), 123.2
127.6 (C-400), 128.5 (C-300),
133.4 (C-10),
135,6 (C-7),
H-600). 13C NMR (100 MHz; CD3OD) :
d
40.1 (C-3),
121.0 (C-6),
126.6 (C-8),
128.9 (C-5),
158.4(C-40).
7,8-Benzo-40-hydroxy-4-imidazolylflavan (2c) was obtained in
d
d
(C-2),
d
d
116.3 (C-30 and C-50),
126.3 (C-500),
128.7 (C-20 and C-60),
151.6 (C-8a),
d
d
d
(C-600),
d
d
d
d
d
d
d
d
d
References
a 46% yield, as a white amorphous solid; mp 180 ꢀC.UV in MeOH
lmax
:
221, 241, 283, 323 nm. HRMS (ESIþ) calculated for
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16 (2008) 1474e1480.
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(2007) 124e130.
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6047e6052.
C22H19N2O2 (M þ H)þ 343.1447, found 343.1444.
2,4-cis isomer : 1H NMR (400 MHz; DMSO-d6):
(2H, m, H-3ax and H-3eq),
d
2.59 to
d
d
2.76
6.05
d
5.46 (1H, br d, J ¼ 10.3 Hz, H-2),
(1H, dd, J ¼ 6.2 and 11.2 Hz, H-4),
d
6.64 (1H, d, J ¼ 8.6 Hz, H-5),
d
d
6.86 (2H, d, J ¼ 8.5 Hz, H-30 and H-50),
d
6.95 (1H, br s, H-4imid),
7.46 (2H, d,
7.60 (2H, m, H-400 and H-500),
7.91 (1H, br s, H-2imid), 8.16
9.49 (1H, br s, 4eOH). 13C NMR
52.5 (C-4), 77.1 (C-2), 115.3
118.3 (C-4imid), 120.3 (C-6),
124.5 (C-8), 126.8
125.9 (C-500),
127,8 (C-20 and C-60),
128.9 (C-5imid),
133.5 (C-7), 137.1 (C-2imid), 149.7 (C-8a), 157.3
7.23 (1H, br s, H-5imid),
d
7.39 (1H, d, J ¼ 8.6 Hz, H-6),
d
J ¼ 8.5 Hz, H-20 and H-60),
d 7.49 to d
d
7.88 (1H, br d, J ¼ 7.6 Hz, H-300),
d
d
(1H, br d, J ¼ 7.9 Hz, H-600),
d
(100 MHz, DMSO-d6):
d
36.7 (C-3),
116.2 (C-4a),
123 0.7 (C-5),
d
d
d
(C-30 and C-50),
121.6 (C-600),
(C-400), 127.5 (C-300),
130.4 (C-10),
(C-40).
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
2,4-trans isomer : 1H NMR (400 MHz; DMSO-d6) :
2.59 to 2.76 (1H, m, H-3ax),
5.66 (1H, br t,
d 2.45 (1H,
dt, J ¼ 2.6 and 14.6 Hz, H-3eq),
d
d
d
5.19 ( 1H, dd, J ¼ 1.9 and 11.1 Hz, H-2),
d
J ¼ 3.8 Hz, H-4),
d
6.81 (2H, d, J ¼ 8.5 Hz, H-30 and H-50),
d 6.97
(1H, br s, H-4imid),
d
7.14 (1H, d, J ¼ 8.5 Hz, H-5),
d
7.23 (1H, br s,
7.49 to 7.60
7,82 (1H,
9.47
H-5imid),
d
7.32 (2H, d, J ¼ 8.5 Hz, H-20 and H-60),
d
d
(3H, m, H-400, H-500and H-6),
d 7,71 (1H, br s, H-2imid), d
br d, J ¼ 8.0 Hz, H-300),
d
8.09 (1H, br d, J ¼ 8.3 Hz, H-600),
d
(1H, br s, 4eOH). 13C NMR (100 MHz; DMSO-d6) :
d 36.5 (C-3),
d
d
d
50.1 (C-4),
119.0 (C-4imid),
125.8 (C-500),
126.7 (C-5),
128.5 (C-5imid),
150.3 (C-8a),
d
73.2 (C-2),
120.0 (C-6),
127.1 (C-400),
130.1(C-10),
157.4 (C-40).
d
112.7 (C-4a), 115.2 (C-30 and C-50),
121.7 (C-600),
124.2 (C-8),
127.6 (C-300),
127.7
133.9 (C-7),
d
d
d
d
d
d
d
(C-20 and C-60),
137.7 (C-2imid),
d
d
d
[23] W. Wouters, R. van Ginckel, M. Krekels, C. Bowden, R. De Coster, J. Steroid
Biochem, Mol. Biol. 44 (1993) 617e621.
d
d
d